阿霉素耐药细胞外泌体通过传递耐药性影响骨肉瘤细胞增殖和迁移的实验研究  

Effects of adriamycin resistance cell-derived exosomes on the proliferation and migration of osteosarcoma cells through drug resistance transmission

作　　者：孙超[1] 冯卫[1] 张丽华[1] 孟晨阳 薛慧琴[1] 赵伟[2] 王玉鑫[2] 王紫横 孙亮[1] 郭世炳[2] Sun Chao;Feng Wei;Zhang Lihua;Meng Chenyang;Xue Huiqin;Zhao Wei;Wang Yuxin;Wang Ziheng;Sun Liang;Guo Shibing(Department of Orthopaedic Surgery,the Second Affiliated Hospital of Inner Mongolia Medical University,Huhhot 010020,China;Department of Bone Oncology,the Second Affiliated Hospital of Inner Mongolia Medical University,Huhhot 010020,China)

机构地区：[1]内蒙古医科大学第二附属医院骨外科,呼和浩特010020 [2]内蒙古医科大学第二附属医院骨肿瘤科,呼和浩特010020

出　　处：《中华骨科杂志》2023年第10期645-658,共14页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金(81960484)。

摘　　要：目的探讨骨肉瘤阿霉素耐药细胞外泌体与MDR1、miRNAs的相关性及作用机制。方法选取骨肉瘤MG63和U2OS细胞株构建阿霉素耐药株,通过MTT法检测耐药和敏感株的50%抑制浓度(half maximal inhibitory concentration,IC50),于15~120 min中间隔15 min观察一次荧光染色检测荧光强度的变化,RT-PCR和Western Blot检测MDR1的mRNA和P-gp蛋白表达水平等验证骨肉瘤细胞耐药性;通过粒径分析及Western Bolt检测鉴定外泌体。观察PKH26标记的阿霉素耐药细胞外泌体的胞吞作用,并采用MTT法和细胞划痕实验检测阿霉素耐药细胞外泌体与骨肉瘤细胞共培养后细胞的增殖水平、迁移能力及耐药性的变化。通过骨肉瘤源性外泌体测序及生物信息分析、RT-PCR验证miRNAs在骨肉瘤敏感和耐药细胞中的mRNA差异表达水平。观察MG63和U2OS中正常骨肉瘤细胞组与耐药细胞组、耐药细胞+正常外泌体组、耐药细胞+耐药外泌体组成瘤裸鼠肿瘤生长情况,血清外泌体鉴定及其miR-21-5p的mRNA表达水平,RT-PCR、Western Blot和免疫组化检测肿瘤组织中MDR1的mRNA和miR-21-5p蛋白的表达水平。结果MG63和U2OS中阿霉素耐药株IC50(11.81、9.33μg/ml)高于正常株(2.21、0.93μg/ml),荧光强度减弱速度高于正常株,MDR1的mRNA和P-gp的蛋白表达水平(2.15±0.10、2.127±0.12;0.92±0.11、0.73±0.10)均高于正常株(1.12±0.16,1.02±0.11;0.46±0.03、0.30±0.04),差异均有统计学意义(P<0.05)。耐药外泌体分别与正常株共培养时可通过胞吞作用进入骨肉瘤细胞内集中分布于细胞质。骨肉瘤细胞与耐药外泌体共培养,分别以2、4、6、8μg/ml的阿霉素浓度处理后,耐药组增殖水平显著上升,耐药组迁移能力(54.20±9.32,19.24±2.88;76.40±5.41,30.26±4.87)较正常组(35.947±3.922,6.72±3.546;51.217±5.546,19.31±1.930)均高,差异有统计学意义(P<0.05)。选择外泌体测序和生信分析中10种上调明显的miRNAs,进行RT-PCR验证其在正常和耐�Objective To explore the relationship and underlying mechanism between exosomes derived from doxorubicin-resistant osteosarcoma cells and MDR1 and miRNAs.Methods MG63 and U2OS cell lines were selected to construct doxorubicin-resistant strains,and the 50%inhibitory concentration(half maximal inhibitory concentration,IC50)of drug-resistant and sensitive strains was detected by MTT,and fluorescence staining was performed at intervals of 15 min between 15 and 120 min to detect the change of fluorescence intensity.RT-PCR and Western Blot were used to detect the expression levels of MDR1 P-gp to verify the drug resistance of osteosarcoma cells.Exosomes were identified by particle size analysis and Western Bolt detection.The endocytosis of PKH26-labeled exosomes from doxorubicin-resistant cells was observed,and the proliferation level and migration of exosomes from doxorubicin-resistant cells co-cultured with osteosarcoma cells were detected by MTT assay and cell scratch assay.The differential expression levels of miRNAs in osteosarcoma-sensitive and drug-resistant cells were verified by sequencing and bioinformatics analysis and RT-PCR assay.Tumor growth,serum exosome identification and mRNA expression level of miR-21-5p in tumor-bearing nude mice between normal osteosarcoma cell group and drug-resistant group,drug-resistant+normal exosome group,drug-resistant+drug-resistant+drug-resistant exosome group were observed.MDR1 expression level in tumor tissue was detected by RT-PCR,Western Blot and immunohistochemistry.Results The IC50 of two adriamycin resistant strains were 2.21 vs.11.81μg/ml and 0.93 vs.11.81μg/ml,respectively,and the fluorescence intensity decreased faster than that of normal strains.The relative mRNA expression levels of MDR1 in two cell lines were normal 1.12±0.16,1.02±0.11 and drug-resistant 2.15±0.10,2.127±0.12,respectively.The relative protein expression of P-gp was normal 0.92±0.11,0.73±0.10 and drug-resistant 0.46±0.03,0.30±0.04,the differences were statistically significant(P<0.05).Dr

关 键 词：骨肉瘤 外泌体 基因 MDR 微RNAS 多柔比星 

分 类 号：R738.1[医药卫生—肿瘤]